Southern Blot for the Identification of Transgene Copy Number

Genomic DNA of potato was isolated from 2 g of leaf tissue from both transgenic and non-transformed control plants by Plant genomic DNA Extraction Kit (TaKaRa), and then 20 µg of genomic DNA was used to perform restriction digestion with HindIII at 37 °C for 16 h. HindIII-digested DNA was separated on a 0.8% agarose gel and the blotted membrane was hybridised with DIG-labelled specific probe, complementary to the sequence of the transgene amiR-159P^{25−HCPro−Brp1} prepared by PCR DIG Probe Synthesis Kit (Roche). Southern blot was performed according to DIG Application Manual for Filter Hybridisation (Roche).